PROJECT SUMMARY Adhesion of cells to one another and the extracellular matrix is a fundamental characteristic of all multicellular organisms. Recent work has shown that mechanical force applied to cell adhesion receptors, including the cadherins and integrins, can affect the activities of Rho family GTPases, thereby influencing the organization of the cytoskeleton and the stiffness of cells. This grant is aimed at understanding how signals from cadherins produce the cytoskeletal rearrangements necessary for cell stiffening, with an emphasis on understanding where the cell derives the energy required to support cell stiffening. In our preliminary studies, we discovered that application of force to E-cadherin stimulates glucose uptake and increases glucose transporters on the plasma membrane. Using molecular, biochemical and cell biological approaches, Aim 1 will define the molecular mechanism for how E-cadherin stimulates glucose uptake. Aim 2 will determine how increased glucose uptake facilitates the cytoskeletal rearrangements required for stiffening. Here we aim to identify the RhoA activator and signaling pathways involved. Using magnetic tweezers and beads coated with the extracellular domain of E-cadherin we will determine how force-induced glucose uptake leads to the strengthening of cadherin-mediated adhesions. The final aim will investigate how the force-induced glucose impacts the actin cytoskeleton and its regulation of cell growth and metastasis in vivo. Mouse models of breast cancer will be employed to explore the consequence that promoting E-cadherin mediated force transmission has on disease. When the work in this proposal is complete, we expect to establish a new paradigm for how glucose uptake is stimulated by force and facilitates cell stiffening. This paradigm can be applied to better understand force transmission by other proteins and will lay the groundwork for understanding if and how E- cadherin force transmission protects against the development of cancer.